JPH0222236A - Monoclonal antibody as signal for directing sensitized effector cell to tumor region and use of conjugate thereof - Google Patents

Abstract

PURPOSE: To obtain a composition containing a sensitized effector cell and an antigen molecule which contains an antibody or its conjugation product capable of binding to a target cell, and useful for exterminating the target cell selected in a mammalian host. CONSTITUTION: An effector cell (e.g. a cell-imparting T-cell) or its precursor is extracted from a man or animal host, and subsequently cultured together with both an effector cell growth factor (e.g. a lymphokine such as IL-2) and the first antigen molecule to produce a sensitized effector cell. The sensitized effector cell is combined with the second antigen molecule containing an antibody or its fragment capable of binding to the target cell and having at least one component common to the first antigen to produce the composition. When the composition is administered into a host, the second antigen molecule is localized at the target cell in the living body, and the sensitized effector cell recognizes the second antigen molecule to specifically direct the cell-impairing effect to the target cell in the host, thereby exterminating the target cell.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention provides methods by which the immune system can be manipulated to treat cancer. Methods are provided for specifically targeting cytotoxic effector cells to target tumor cells in vivo. Effector cells (which can be T-cells) can be sensitized in vivo.

[Background technology]

Current methods commonly used to treat cancer include surgery, radiation therapy, and chemotherapy, with immunotherapy emerging as the fourth major treatment modality. Chemotherapy, control and surgical therapy have some risks and side effects due to their limited specificity for tumor cells, so there is no substitute for less toxic and more effective methods of cancer immunotherapy. Research has been carried out by scientists for a long time. However, several problems exist in the clinical context. First, some tumor antigens are not sufficiently "immunogenic" to mount a clinically meaningful response; furthermore, the number of activated lymphocytes available or their activity level is limited. It may not be sufficient to successfully suppress large tumor burdens. These two major limitations are problems that have hampered efforts to design effective immunotherapeutic systems today.

Discovery of monoclonal antibody technology (Kohler and Mi
Stein, 1975), it is now possible to generate antibodies against tumor-associated antigens in large quantities and in a homogeneous form. Furthermore, methods are available today to expand and activate immune effector cells that can elicit antitumor effects both in animal models and in humans (M.A., Cheever et al., Journal
of Ima+unolo+1981;T,J
, Eberlein et al., Journal of Ex
erimental medicine, 1982
;S, A, Rosenberg, New En 1
and Journal of Medicine.

1985). Much of this research has been made possible through the discovery of lymphokines, which are metsenger molecules that stimulate or influence immune effector cells. In particular, interleukin-2 (IL-2) is a lymphokine-activated killer (LAK) cell that stimulates the proliferation and activity of a subpopulation of T-cells in cell culture and has anti-tumor effects. (D, A, Morgan et al., 5ci
ence, 1986; [i, A.

Griffiths et al., Journal of Exer.
in+mental Medicine.

1982).

Some terms and concepts related to this invention require the following definitions.

1. Goo Mata Keimen Danbo: can induce a specific immune response under appropriate conditions, and the products of the response, i.e. specific antibodies or specifically sensitized T-lymphocytes or A substance that can react with both.

2. Five stops: ! An immunoglobulin (a form of protein) molecule that is synthesized by cells of the J. lymphoma lineage and has a specific amino acid sequence and therefore interacts with the antigen that induced its synthesis or with antigens closely related thereto.

3. Monoclonal Antibodies derived from cell clones (Today, most monoclonal antibodies are Koh
Monoclonal antibodies, although resulting from fusion leading to the formation of hybridoma cells as described by Ler and Millstein, can also be developed by genetic engineering or potentially by other synthetic methods). Monoclonal antibodies can be used as whole monoclonal antibodies or fragments thereof.

4. Habten: has a chemical configuration that allows it to interact with a specific binding group on an antibody or with a recognition site on a T-lymphocyte, but unlike an antigenic determinant, it does not itself Certain protein-free substances that do not elicit an immune response (eg, a detectable T-cell response or the production of detectable amounts of antibodies). When linked to a carrier protein, it elicits an immune response.

5. Yeongdanto nib: Antigenic determinant (one antigen may have multiple epitopes) 6. Yul main safue:
Soluble protein mediator-07, mitogen: released by certain lymphocytes and capable of limiting other cell-mediated immune functions, such as lymphocyte transformation, macrophage activation, or cytotoxicity to other cells. Substances that induce mitosis and cell transformation, especially lymphocyte transformation.

Unlike heterologous antisera, monoclonal antibodies can be produced in large quantities and obtained in homogeneous batches for use in mammals, including humans. Monoclonal antibodies have been shown to:

1. Collects in tumor tissue after intravenous injection (R,K.

01dha-et al., Journal of Cl1nic
al 0ncoio + 1984).

2. Shows selective biodistribution in tumors with some non-specific uptake into normal cells (S,M).

arson et al., Journal of C11
nical Inversion.

1983; P.G., Abrams et al., A.A.
Reilsteld and S.

Se11w1. , Monoclonal Antibo
dies and Cancer﹃μ7肚, 198
5).

3. When antibodies capable of enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) are used, they induce tumor regression in some patients.

Most antibodies used to support ADCC were of the IgG-3 subclass. Since ADCC-inducing activity is primarily associated with the FcTiI region, these antibodies have been used as whole molecules rather than as fragments. ADCC-associated tumor regression was inconsistent and occurred in a minority of treated patients (A, N, Ho
ughLon, supra). Furthermore, the requirement to use whole antibodies reduces flexibility in the use of fragments that have different biodistribution properties and offer certain advantages in tumor localization. Therefore, there is a clear need to develop methods to enhance the ability of antibodies to produce ADCC (and subsequent tumor regression) in a more consistent and reproducible manner.

A by independently stimulating effector cells in vivo or by administration of lymphokines such as IL-2.
Research into the use of lymphokines to aid DCC is currently underway. However, this is a non-specific method aimed at activating effector cells in general.

Adoptive immunotherapy using autologous effector cells (adoptive ia+s+uno
therapy) has received much attention based on the observation that anti-tumor effects occur in humans (S, A,
Rosenberg et al., New En 1 and J.
our own of Medicine, 1985 and 1987; W,) 1. West et al., New E
n 1 and Journal ofMedicine
, 1987). It is clear that tumor regression occurs in both animal models and humans following injection of L AL cells and subsequent injection of Roux2. These cells are first obtained from a donor (patient) by cytophniresis (cytafniresis).
apheresis) and cultured in vitro in the presence of IL-2, thus resulting in activated killer cells. These cells are expanded by tissue culture and when returned to the patient, administration of IL-2 further enhances their continued expansion in vivo. This manipulation has been shown to result in tumor regression in various systems. Examples of animal cell models include murine FB
L~3 Leukemia (M, A, Cheever, Jour
nal of In+munol.

1981; T, L., Eberlein et al., Jou
rnal of ex erimentalMedic
ine, 1982), murine EL-4(G) lymphoma (M,^, Cheever, supra), and murine m
eth-A sarcoma (M, J, Berendt et al., Jour
nal of Ex erimental Media
ns, 1980). Human tumors for which efficacy has been observed include renal cell carcinoma, heterogeneous melanoma, and in some cases other types (S, A, Rosenberg et al.
New En 1 and Journal for Me
dicine, 1986 and 1987; W, H,
West et al., New En 1 and Journal
ofMedicine, 1987; J, R, Du
rant, New En 1 and Journal
of Medicine, 1987). In this approach, the activation event for killer cells is non-specific and consists of in vitro exposure to IL-2 in cell culture. Since the cytotoxicity of these cells appears to be selectively exerted in tumors, immunological markers naturally expressed on tumor cells may attract LAK cells to the tumor site, or perhaps <LAL It is expected that IL-2 administered after injection of cells will have some effect enhancing immunogenicity/antigen expression on the tumor or contributing to cytotoxicity. There is no established method to specifically target LAK cells to tumors. However, tumor-infiltrating cells will exhibit substantially more effective cytotoxicity if they are obtained from tumor biopsies and expanded in vitro (S, 8, Rosinbe et al.
RG et al., 5science, 1986), the efficacy of such adoptive immunotherapy could potentially be increased if this cell population could be obtained and cloned.

In an effort to explore the selectivity of cells activated by lymphokines, researchers have compared lymphocytes in culture to irradiated cells from experimental tumors in animal models. L
-2, thereby attempting in vitro immunization (S, G11lis, Journal of Ex
erimental medicine, 1978
; M.A., Cheever.

Journal of! ++usunolo 11
981) The expanded effector cell population is particularly effective against immunized tumors when tested for cytotoxicity in both in vitro and in vivo experiments for the treatment of tumor-bearing animals and Less effective against other tumors used as controls. Ta. This phenomenon was confirmed by similar experiments by other researchers (J, L
, 5trausser et al., Journal of Iv.
sunol.

1981: T. C. Eberlein et al., Jour
nal of the National Cancer
1 institution+ 1982) *These effector cells have a T-cell phenotype. Therefore, early ex vivo exposure to tumor antigens may result in restricted (con
clones of cytotoxic T-cells can be generated, and these cells selectively exhibit their effects on the specific tumor used for their immunization, but not on other tumors. It seems to demonstrate. It may be difficult to apply the above techniques to human tumors because the tumor cells or antigens from a given patient are not available in sufficient quantities or sufficiently immunogenic. There would be a risk in implementing a therapy that would potentially result in reintroduction into the patient of tumor cells previously obtained and used for in vitro immunization of effector cells. Furthermore, some tumor antigens may not be sufficiently "immunogenic" to initiate a clinically meaningful response. moreover,
The number of activated effector cells available or their activity level may not be sufficient to successfully suppress large tumor burdens.

Therefore, the various approaches to immunotherapy described above suffer from heterogeneity of results, lack of wide clinical use in humans, II.
Problems include poor immunogenicity of some types of IJX and an inability to generate sufficient activity and/or quantity of effector cells for effective treatment. There is a need for immunotherapy that circumvents such difficulties.

[Summary of the invention]

The present invention provides a method for eradicating target cells in a human or animal host, the method comprising: a) extracting effector cells (or precursors thereof) from the human or animal host; b) said extracted effector cells. in vitro with both an effector cell growth factor and a first immunogenic molecule, thereby producing sensitized effector cells; C) comprising an antibody or antibody fragment that binds to said target cell; and administering to said host a second immunogen molecule having at least one component in common with said first immunogen molecule; and d) administering to said host the sensitized effector cells generated in step (b). ;consists of stages.

Preferably, effector cell growth factors are also administered to the host. The effector cell growth factor can be a lymphokine such as interleukin-2 (IL-2). In one embodiment of the invention, administration of effector cell growth factors to the host begins after step (C) and continues at least until completion of step (d). Alternatively, administration of effector cell growth factors to the patient can begin at other times, such as after step (d).

The step for extracting effector cells from the host is to subject the host to leucapheresis (1 eu) once or multiple times a day.
kapheresis) (e.g., one or more times every 24 hours). In one embodiment of the invention, the effector cells are cytotoxic T-cells.

Sensitized effector cells are generated by incubating the effector cells in vitro with both an effector cell growth factor (eg, a lymphokine such as C2) and a first immunogen molecule. The sensitized effector cells recognize the first immunogen molecule or its components.

The first immunogen molecule may be the same as the second immunogen molecule or different, but these two immunogen molecules must have at least one component in common. The second immunogenic molecule comprises an antibody that binds to the desired target cell and therefore localizes on the target cell in vivo after administration to the host.

A second immunogen molecule (or a common component thereof) that is located on the target cell in vivo by the effector cells sensitized in vitro in step (b) to recognize the first immunogen particle.
The first immunogen molecule and the second immunogen molecule are selected such that they also recognize the immunogen molecule. Thus, the cytotoxic effects of sensitized effector cells are specifically directed against target cells in the host.

A cocktail of immunogenic molecules can be used in steps (b) and (c). That is, generating a population of effector cells sensitized to recognize multiple different immunogens, and transplanting multiple "engineered" antigens onto the target cells (perhaps using two or more target-specific antibodies). ). The use of cocktails is useful when the target tumor cells comprise a heterogeneous population of tumor cells, or when at least one of the engineered antigens (e.g., histocompatibility antigens) is non-self in the intended patient. To guarantee, it is advantageous.

Target cells are unwanted cells that are to be eradicated within the host;
For example, cancer cells. The target cells are cancer cells. case,
The antibody component of the second immunogen molecule (and in certain embodiments of the invention, the first immunogen molecule) can be a monoclonal antibody that binds to the target cancer cell.

Another method for eradicating target cells in a human or mammal comprises administering to the host an immunogenic molecule comprising an antibody-immunogen conjugate that binds to the target cell, wherein Binding of the immunogenic molecule to a target cell induces a cytotoxic effector cell response directed against the target cell. Therefore, if an immunogenic molecule is capable of iARing a therapeutically effective cytotoxic effector cell response in vivo without a presensitization step, in some cases leucapheres is and in vitro sensitization would not be necessary.

Additionally, the present invention provides a variety of immunoconjugates comprising an antibody that binds to a desired target site in a human or mammalian host and a polypeptide that is foreign to the host. The polypeptide is attached to the antibody directly or via a linker or spacer molecule. Such immunoconjugates include conjugates in which class ■ or ■ histocompatibility antigens (non-self or non-human) or fragments thereof are conjugated to target-specific antibodies. Class I or ■
A histocompatibility fragment can be an alloantigen domain. Immunoconjugates of the invention can be generated by coupling peptides that are cytotoxic T-cell epitopes to target-specific antibodies.

[Specific description of the invention]

The present invention provides methods for eradicating target cells in vivo. This method uses in vitro sensitization of effector cells (eg, cytotoxic T-cells or natural killer cells) toward immunogenic molecules.

The immunogen molecule is then bound to the target cells in the patient by using antibodies specific for the target cells. The sensitized effector cells are then administered to the patient, and these cytotoxic effects are specifically directed against target cells carrying the immunogenic molecules used to sensitize the effector cells in vitro. Directed.

As mentioned above in the “Background Art” section, tumor-associated antigen (T
It is now clear that in vitro exposure to AA) and IL-2 can cause apparent effector cells (usually T-cell-derived lymphocytes) to develop certain tumor-associated antigens (T^8). It is. These cytotoxic cells can then produce antitumor effects both in vitro and in vivo (S, G11lis et al., Jo
Urnal of Ex erimental Medi
cine, 1978; M. A. Cheever
etc., Journal of HaKakeRubao■+ 1981;
T, J, Eberlein et al., Journalof
the National Cancer In5t
itute+ 1982; J, L.

5trausser et al., Journal of l5u
sunolo + 1981).

These immune effector cells are then programmed to recognize and selectively exert cytotoxicity against tumor cells bearing selected antigens. Application of this concept will have a substantial impact on cancer treatment. However, several problems exist in the clinical environment. First, some tumor antigens may not be sufficiently "immunogenic" to mount a clinically significant response.

Furthermore, the number of activated effector cells available or their activity level may not be sufficient to successfully suppress large tumor burdens. The present invention is designed to overcome these problems as follows.

1. Using a target cell-specific monoclonal antibody that is itself highly immunogenic, or comprising a highly immunogenic polypeptide conjugated to an immunoconjugate or monoclonal antibody, such as a monoclonal antibody-habten conjugate. Strong immunogenicity is ensured by forming immunoconjugates.

These monoclonal antibodies or conjugates thereof, such as antibody-habten conjugates, bind to target cells and function as "processed target antigens" after they are located on a target cell (eg, a tumor cell).

2. Tumor-bearing subjects were treated with Lucafuerize (1eukap)
producing a large number of sensitized and activated cytotoxic effector cells specific for the target cell of interest by (heresing) and from this population cultured in the presence of the antibody or conjugate thereof. restrained (co
The sensitized clones of effector cells (LAK cells or committed T-cells, or potentially other effector cells) are expanded in vitro. This is generally done in the presence of IL-2 or other lymphokines that stimulate effector cell proliferation.

3. First, the antibody or its conjugate is administered to the patient, and after the conjugate is localized to the target cells and expresses the processed antigen, a large number of ex vivo sensitized effector cells are injected to The engineered antigen-bearing target cells are recognized and cytotoxic to them.

That is, monoclonal antibodies serve as carriers to implant a strong antigenic signal into target cells, ensuring that the target cells are fully "immunogenic."

The engineered antigen bound to target cells attracts pre-sensitized effector cells to the target cells in vivo. This method actually constitutes amplification of tumor-associated antigens.

The injected effector cells are co-cultured with this predetermined antigen signal, thereby sensitizing the effector cells to what can be described as a processed tumor antigen. Large numbers of cytotoxic effector cells are produced by ex vivo expansion for injection into a patient, and these cells can be further expanded by the use of lymphokines such as IL-2 administered in vivo.

If the target cell-specific monoclonal antibody is sufficiently immunogenic in terms of sensitization of effector cells, this antibody alone can be used as engineered antigen. Alternatively, highly antigenic molecules such as haptens or certain polypeptides can be conjugated to antibodies and the resulting immune complexes used as engineered antigens. Such immunoconjugates and methods for their preparation will be discussed in detail later.

Target-specific antibodies suitable for use in the present invention include polyclonal antibodies and monoclonal antibodies;
Monoclonal antibodies (MAbs) specific for target cells
) is preferred. A number of monoclonal antibodies that bind to specific types of cells have been developed, including tumor-associated antigens in humans, and methods for producing such antibodies are known. A large number of such M
Abs include anti-TACl or other IL-2 receptor antibodies;
9, 2.27 and NR-Ml-05, 37 for 250 kilodalton human melanoma-associated proteoglycans
-40 kilodaltons - pancarcinoma (pancor)
cinoma) NR-Lu- for glycoproteins
10; and 0 for as yet unidentified cancer-associated antigens.
VIh#' is included. Antibodies derived from genetic or protein engineering can be used as well. Antibodies used in the invention can be intact molecules, fragments thereof, or functional equivalents thereof. Examples of antibody fragments include F(ab')z + Fab' + Fab and F
v fragments, which can be produced by conventional methods.

In summary, the methods of the present invention provide a method for amplifying the antigenicity of target cells, such as am cells, by implanting a strong antigenic signal into the target cells, such as tumor cells, using monoclonal antibody carriers. To enhance host immunocompetency, lymphokines and certain ``engineered''
in vitro (eneered) J antigen signal
effector cells by in vitro exposure
x vivo) operation is performed. After the engineered antigen (comprising a target cell-specific monoclonal antibody) has been administered to the patient and localized to the target cells, cytotoxic effector cells conditioned to recognize said antigen signal are injected, thus The immune response against the tumor is amplified.

According to the method of the invention, effector cells are extracted from a human or mammalian host. Effector cells are "sensitized" to recognize a particular antigen and have a cytotoxic effect on target cells bearing that antigen.
ze) Cells that can be treated with J.

The effector cells used are “cov-restricted” (cov).
++1tted) cells described as "T-cells",
and various cytotoxic cells with other names, such as LAK cells, natural killer (NK) cells, and other lymphocytes and macrophages that can potentially be sensitized and similarly manipulated. In one embodiment of the invention, the effector cells are cytotoxic T-IJ lymphocytes, also referred to as cytotoxic T-cells.

effector cells in the host (e.g. the patient to be treated)
It is extracted from as follows. That is, cells are collected from the patient and effector cells of interest are separated from all other cell types and co-extracted biological material by any suitable method. Routine cell harvesting or cytopheresis (
For example, Ieukapheresi
s) ) method can be used and this effector cell extraction method can be repeated to obtain large amounts of effector cells.

For example, the host can be incubated for one day or even longer (e.g. 5 days to collect large numbers of effector cells, e.g. T-cells).
Leukap eresis once a day
heresis) method.

The extracted effector cells are incubated with both an effector cell growth factor and a first immunogen molecule to produce sensitized effector cells. The effector cell growth factors used vary according to the type of effector cells used and enhance cell activation and stimulate proliferation of cultured cells such that sensitized cytotoxic effector cells are produced. can be any growth factor that Suitable growth factors include monokl for monocytes and lymphokines for lymphocytes such as T-lymphocytes (e.g. fL).
-2).

The generation of numerous lymphokine-activated killer cells for clinical utility has been well described in the literature (Rosenbe et al.
rg, S, ^0, etc., New En 1 and Jou
rnol ofMedicine+ 1985;Mu
ul, L, M,, Journal of fmun.

1 wa, Methods, 1986; West
, W, H, etc., Stone 7 Journal of Med
As previously discussed, the initial exposure of effector cells to target antigens in animal models has also been deeply described (Gillis, et al., Journal of Exerimen La1M).
edicine+ 1987; Cheever,
M, 8, etc., Journal offmuno
lo■, 1981). Variations on these described methods are suitable for use in the method of the invention.

The first immunogen molecule (with which the effector cells are incubated) may be the same as the second immunogen molecule (with which the patient is administered), or it may be different. However, the two types of immunogen molecules have at least one component in common, such that effector cells sensitized in vitro in the presence of the second immunogen molecule The molecule must be recognized in vivo.

If an antibody (preferably a monoclonal antibody) specific for the target cell of interest is sufficiently immunogenic, it can be used alone as the first and second immunogenic molecule. As used herein, "immunogen" (or immunogenic or immunogenic; isn+unogen
The term ic) means capable of inducing sensitization of effector cells.

That is, effector cells can be sensitized to recognize the immunogenic molecule.

Certain antibody production methods result in antibodies with increased immunogenicity. For example, genetically engineered antibodies produced in yeast incorporate mannose molecules and are glycosylated, resulting in antigenic mannan structures. The antibody of
Even without the addition of other structures to the antibody, it will have sufficient antigenicity to be used as the first antigenic molecule in the present invention.

Methods for producing antibodies have been extensively described in the prior art and need not be further described here (Kohler and Milstein, 1975;
Il, Zole I, 19B? ), whole antigen or antigen fragment (Fab.

Fab' + F(ab')z, or smaller fragments produced by conventional methods] can be used if appropriate, in which case these fragments are immunoreactive (im
lIIunoreactivity) and immunogenicity (i
The immunogenicity of a particular antibody can be tested in appropriate routine cytotoxicity assays or in animal studies. Accordingly, such in vitro assays or studies can be used to determine whether a particular antibody is sufficiently immunogenic for use as a first immunogenic molecule.

Alternatively, various compounds can be added to antibodies to increase their immunogenicity. Types of compounds that can be attached to antibodies for this purpose include haptens and polypeptides that are "foreign" to the intended patient (e.g., antigens, mitogens, other foreign proteins, and fragments thereof, or cytotoxic agents). There are peptides that activate sexual T-cells).

In some cases, attachment of a compound to an antibody via a spacer (ie, a linker molecule that functions to physically separate the compound from the antibody) will enhance immunogenicity.

The definition of hapten is as described above. Hapten groups that can be added to antibodies according to the present invention include benzoic acid group,
the nitrophenol group, other small molecules such as, but not limited to, acetic acid and its derivatives, benicilenic acid and its derivatives, sulfanilic acid derivatives, hexosamines, and possibly ribonucleotides or ribonucleosides, and isocyanates and isothiocyanates. .

These are examples of various molecules that can be used as hapten components for the purposes of the present invention, and other hapten groups are suitable as well. Certain larger hapten molecules are more immunogenic than smaller hapten molecules and can induce more effective T-cell responses.

The scientific configurations of some of these hapten molecules are shown below.

11, C-C-OH Dinitrophenol To1nilophenol - Sulfnir C-10 (CHHz)s OOH oon -Hydroxyphenyl II OOH CH.

Antibodies can also be conjugated with various antigens to enhance the antigenicity of the antibody molecule. These include polypeptides that are "foreign" to the intended host, ie, recognized as foreign (non-self) polypeptides by the host's immune system. Such polypeptides for use in human patients include hydrolyzed dextran, flagellin, fragments of keyhole limpet hemocyanin, histocompatibility antigens, bacterial cell surface antigens, viral coat proteins, and fragments thereof. It will be done. Smaller molecules (ie, polypeptide fragments) are generally preferred in an effort to avoid interfering with the biodistribution of antibodies (or antibody fragments).

In vitro polypeptide synthesis, protein engineering, and recombinant DNA
Synthetic polypeptides produced by such methods can be added to antibodies to enhance the antigenicity of the antibody molecule.

T-cell mitogens, such as the protein pokeweed antiviral protein (PA
P) and phytohemagglutinin (PHA) can also be used. Among these, PAP has a low molecular weight (32
,000) for PHA (molecular weight 140.000)
More preferred.

Polypeptides that have no similar counterpart in humans are more immunogenic and are therefore preferred for use in the present invention. Such polypeptides include bacterial cell surface proteins,
Particularly included are viral coat proteins and fragments thereof. Several such proteins, known to have human T-cell counterparts, have been characterized. For example, the amino acid sequence of influenza hemagglutinin is known, and the protein (or antigenic fragment thereof) can be purified or synthetically produced for use in the present invention.

The effector cells used to kill tumor cells are 2
CLan1er and Ph1ll belonging to two broad classes
ips, button munolo Toda, 7.132
(1986)).

The first class is a subset of natural killer (NK) cells and cytonegative T-cells, which are involved in the major histocompatibility complex (major histocompatibility complex).
tycomple; M) IC) that acts on target cells without restriction (i.e. M) for its activity;
(does not require HC class I protein). The antigens directly conjugated to antibodies are designed to activate these cells. Various antigens have been proposed to activate natural killer cells. Because of their structural requirements or the NK they bind
This is because little is known about cell surface receptors on the cell surface. The second class includes MHC-restricted cytotoxic T-cells. They play a central role in the immune response to allogeneic cells infected by foreign organisms and are thought to be involved in immune system surveillance against tumor cells. However, some malignant cells are thought to evade immune system surveillance due to the lack of immunogenicity of tumor-associated antigens and due to various suppressive mechanisms present in tumor-bearing animals (Berendt MJ and North RF, J , Kaisei-1 Blood, 69(1
980) ). Many attempts have been made to enhance tumor antigenicity by linking antigenic determinants to IIl tumor cells or by inducing antigenic variants [
Flood, PM et al., J.

Immunol, 138.3573-9 (1987
) and references therein].

Some tumors, such as certain types of leukemia, bladder tumors,
Sarcoma, or methylcholanthlena
For anthrena)-induced tumors, tumor-derived clones metastasize more easily if they lack MHC antigen expression (Festenatein II and Garrido, F., Nature, 322+
502 (1986)], other tumors in which MHC antigen expression is associated with tumor growth include mouse lung cancer and possibly human small cell lung cancer, as well as other tumors (13ahl
er, DH et al., PNAS, 84°4562-6 (
198) and references therein).

However, there are numerous examples where autologous MHC antigen expression enhances tumor growth, such as Maloney virus-induced YAC-1111JJ% cells, and solid tumors such as breast cancer, colorectal cancer or melanoma. There appears to be extreme heterogeneity in MHC antigen expression in the individual cells found in
in and Garrido, 1986).

All of these studies have focused on self-MHC antigen expression. MH on some tumors, presumably avoiding the risk of the presence of MNC autoantigens on the surface of other tumor cells.
Novel approaches to immunotherapy that can prevent defects in C antigen expression include the use of immunoconjugates comprising non-self Mt (C antigen) bound to tumor-specific antibodies, according to the present invention. This is an intentional labeling.It is abnormal (
aberran t) MHC-like antigens are known to result in the rejection of Rous sarcoma virus-induced tumors that do not express normal self-class IMHc antigens (Peste et al.
nsLein and Garrido, 1986). Similarly, alloantigens reactive with the MHC antigen itself
Adoptive transfer of T-cells has been used successfully (MerediLh RF and Okonewich).
JP, Trans 1antation, ,IM
+378-8C1983). This approach should utilize classical immune system surveillance and rejection of foreign cells;
This is recognized by the presence on their surface of non-self class I or nMHC antigens. These responses can be e.g.
In graft-versus-host disease (R, 5torb.

”Graft vs-bost disear
e after marroin transplan
tation, Ee mutual)■↓anLatio
n: A roaches t.

Graft R Merryman HT, I
)^fan R, Li5s.

New York, 1986), or may be important in transplant rejection in HLA-mismatched individuals.

In one embodiment of the invention, the heavy (α) chain of a non-self or non-human MHC class 1 or 1 antigen protein, much of the sequence of which has been elucidated.

Figueroa F and Klein J, Immu.
r++ Ebong L1 鉦+ 1+41-44.1986, or G11es RC and Capra JD, 8dvan
ces in Ia+munolo+37+1
-71+ (see for example 1985) the extramembranous part of M! By conjugating antibodies directed against tumor-specific antigens, cytotoxic T-cells can be directed to recognize 11m71 cells as "foreign" and lyse them. In another approach, alloantigenic sites, ie, cytotoxic T-cell recognition sites on MHC class I or II antigens, can be covalently linked to anti-tumor antibodies. A fragment of IILA-2 that inhibits cytotoxic '7' IJ lymphocyte (CTL) responses has been described (Parham et al., Nature, 325
．． 625.1987) and the three-dimensional structure or sequence of HLA-2A, the best studied HLA antigen (Bjor
kian PJ etc., Nat and sleep↓ vice, 506-51
8.1987), the alloantigenic site has not been precisely defined. Therefore, human or non-human soluble non-
Autologous class I or class II histocompatibility antigens, or fragments containing alloantigenic sites, can be conjugated to anti-tumor antibodies.

In another embodiment of the invention, an immunoconjugate is formed as a fusion protein of an anti-tumor antibody or fragment thereof with a class I or II non-self antigen or an alloantigen fragment of these antigens. Alternatively, soluble class ■ or ■ non-
Self-MMC antigens (i.e., histocompatibility antigens) or alloantigen fragments can be isolated by expression of DNA encoding the sequences, or by proteases or organic chemicals.
It can be formed by fragmentation of C protein.

The use of immunoconjugates containing non-self HLA antigens can induce activation of helper T-cells reactive against HLA antigens. These cells are immunoconjugates or HLA
increase cytotoxic T-cell responses to antigens and further support T-cell responses to other cell surface antigens;
This would enhance the tumoricidal effect desired here (Fujiwara H et al. Ce1lular Ia
+munothera of Cancer, R,L
, Truitt, R9P, Ga1e, M.M.
Bortin, New York, Alan R. Li.
5s Inc, +335-344 True, 1987).

Other classes of immunogens that can be conjugated to anti-tumor antibodies include peptide fragments of foreign immunogenic proteins that are cytotoxic T-cell epitopes. These peptides have been shown to induce proliferation of antigen-specific cytotoxic T-cells and can be conjugated to antibodies either directly or by long spacer arms. An example of a human cytotoxic T-cell epitope from influenza virus nucleoprotein is the peptide 5AAFEDLRVLS.
Includes FIRF and IASNENM AMESSSTLE (Townsend+ A, R, M.

et al., juxtaposition, 44.959-968.1986).

The first immunogen molecule and the second immunogen molecule can be the same, in which case both molecules are immunogenic target-specific monoclonal antibodies, or both molecules are coupled to a target-specific monoclonal antibody. and one of the haptens or foreign polypeptides described above.

In other embodiments of the invention, the first immunogen molecule need not comprise an antibody. For example, the first immunogenic molecule can be one of the foreign polypeptides described above (eg, an antigen, a mitogen, or a fragment thereof). In this case, the second immunogen molecule can be a first immunogen molecule attached to a monoclonal antibody that binds to target cells. Effector cells that have been sensitized in vivo to recognize a first immunogen molecule can also recognize that molecule in vivo and target cells that have the immunogen bound to it via monoclonal antibodies. It exhibits cytotoxicity against.

In a second example, the first immunogenic molecule comprises a hapten attached to one of the aforementioned polypeptides, and in this case the second immunogenic molecule comprises the same hapten attached to a monoclonal antibody that binds to the target cells. Contains a hapten. Effector cells would still recognize the hapten group, but would be expected to be better sensitized if the hapten was on a different protein. Alternatively, such manipulation may prove to be cheaper or more practical (if the hapten is conjugated to albumin and an antibody, the former used in vitro and the latter used in vivo). ).

A number of standard chemical linkage methods can be used to attach various haptens or polypeptides to antibodies. The method by which a compound is attached to an antibody will vary depending on the chemical structure of the compound. Antibodies may contain various functional groups, such as carboxylic acid (COOII), or free amine groups (-Nl
2) and these groups are available for reaction with appropriate functional groups on the compound to attach said compound. Alternatively, antibodies and/or compounds can be derivatized to expose or add additional reactive functional groups. Derivatization can be achieved using a number of linker molecules, such as Pierce Chemical
(Pie L., Inc., Rockford, Illinois).
rce 1986-87. General Cata
log, pages 313-354). Bifunctional linkers having one functional group that reacts with a group on a particular drug and the other group that reacts with an antibody can be used to form the desired immunoconjugate.

Alternatively, derivatization can be accomplished by chemical treatment of the antibody, such as by glycol cleavage of the sugar moiety of the glycoprotein antibody with periodic acid to generate free aldehyde groups. Free aldehyde groups on an antibody can react with free amino or hydrazine groups on a compound to couple the compound to the antibody (see U.S. Pat. No. 4,671.958) on the antibody or antibody fragment. Methods for generating free sulfhydryl groups are known (U.S. Pat. No. 4,659
, 839).

Many methods and linker molecules are known for attaching various compounds, including polypeptides, to proteins such as antibodies. For example, U.S. Patent Nα4.671,958
,k4,659,839 ,No,4.414,148
, Na4.699,784 , Na4,680,33
8, Nα4.569,789, and Na4,590
．． See 071.

The ligation reaction should be performed under conditions mild enough to avoid denaturing or damaging the antibody or polypeptide. The hapten or polypeptide does not dissociate from the antibody in vivo and remains bound (via the antibody) to the target cell for at least a sufficient period of time to induce a cytotoxic T-cell response against the target cell. First, the connection should be stable.

Small hubten molecules such as benzoic acid can be easily conjugated to antibodies in the presence of linking reagents such as ECD 1 (ethyldimethylaminopropylcarpodiimide).

Some examples of how haptens are linked to antibody molecules (or fragments) are as follows.

Antibody Benzoic acid *ECDI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidohydroxyphenylacetic acid, acetic acid, 3-iodo-4-hydroxy-5-nitrophenylacetic acid, and p-sulfanilic acid as an antibody, Conjugation can be achieved by removal similar or comparable to the method described above for benzoic acid. Optimal conditions will vary.

Nitrophenyl derivatives also provide antibodies with the ε of the IJ lysine group.
Linkage can be achieved by interaction with the -NH2 group, the α-N11□ group of the terminal amino acid, or the SH group of cysteine.

for example, Other examples are:

Other examples are:

Para-7minoben7aneflu9ate (7g! T-ruic acid) Diazonium compound anti-atom (having a tyrosycyl residue) More detailed examples of linkage methods are described in Examples 1a and 1b.

Advantageously, effector cell growth factors are administered to the patient in order to enhance the proliferation and activity of sensitized effector cells in vivo. Generally, the effector cell growth factor administered is the same effector cell growth factor that was used to induce effector cells in vitro during the sensitization step. Suitable growth factors will depend on the type of effector cell used and include lymphokines such as IL-2, as described above.

The method of administration of growth factors can vary. For example, several doses can be administered or the infusion can be continued for several days. A suitable method is illustrated in the example below.

In some cases, an "engineered antigen" to be bound (via an antibody) to a target cell is used.
is sufficiently immunogenic to induce a therapeutically effective cytotoxic effector cell response in vivo without sensitizing the extracted effector cells to immunogenic molecules in vivo. Accordingly, other methods for eradicating target cells in a human or mammalian host include administering to the host an immunogen molecule comprising an antibody-immunogen conjugate that binds to the target cells in vivo. The binding of the immunogenic molecule to a target cell in the host induces a cytotoxic effector cell response directed against the target cell.

The immunogen molecule comprises one of said foreign polypeptides linked to a target-specific antibody.

The administration of immunogenic molecules (engineered antigens) targets cells, such as tumor cells, by implanting them with an antigenic signal that induces stronger effector cell responses than the target cells alone (i.e., is highly antigenic). Functions to enhance the host's immune response to target cells. As mentioned above, certain types of 1!1 tumor cells do not have sufficient immunogenicity to induce a strong immune response. For example, various types of cancer cells lack expression of class I histocompatibility antigens, and this is believed to be a mechanism by which these cells evade immune system surveillance.

When certain engineered antigens of sufficient immunogenicity bind to such cells (via antibodies), the host's natural effector cell population responds. For example, if the immunogenic molecule comprises a non-self-histocompatible antigen or a fragment thereof conjugated to an antibody (as described above), the host's natural T.
- Cellular surveillance is expected to result in a cytotoxic T-cell response directed against target cells bearing the engineered antigen. A second example is an immunogenic molecule comprising one of the above-mentioned antigenic polypeptides from a virus or bacteria, conjugated to an antibody. Many patients already have T-cells sensitized to such polypeptides as a result of prior exposure to viruses or bacteria.

Optionally, the methods of the invention include the additional step of extracting effector cells from the host and incubating the extracted cells with lymphokines in vitro to activate them. Lymphokines (e.g. IL-2)
The method of activating effector cells such as T-cells to generate lymphokine-activated killer cells (LA cells) has been previously described. The activated effector cells are then administered to the patient who has been administered the immunogenic molecule. Lymphokines can also be administered to the patient as described above. It is now clear that intravenously administered antibodies or fragments thereof can localize in tumor tissue. Generally speaking, this would be the route of supply. However, changes may be made depending on the actual environment. For example, administration may be intralymphatic or intraperitoneal, as appropriate.

It is expected that therapeutic benefit will be obtained in patients with more intact immune systems (not interfered with by modern chemotherapy or radiation therapy). However, the treatments described in this invention can be applied to patients simultaneously or in combination with other therapies. For example, excision surgery can be performed prior to treatment to limit tumor burden.

Radiation therapy to large tumors given simultaneously can also serve the same purpose. In this case, it would be necessary to cytapherese the patient prior to administering radiotherapy to protect circulating stem cells. After these effector cells have been expanded in culture, controls can be performed after the injection of antibody and before each effector cell dose. In this case, the concept is that LI tumor cells damaged by the control, even if they ultimately prove to be control-resistant, due to their recognition of the pre-supplied "processed antigen". are potentially more susceptible to the cytotoxic effects of immune effector cells attracted to that area.

Target-specific antibodies selected for use in the present invention are rapidly internalized (inter-nal) by target cells.
ize) should not be done. This is because the antibody (or fragment thereof) should be retained on the target cell surface for a sufficient period of time to attract effector cells to the target site.Antibody or antibody conjugate (“engineered antigen”)
If it is internalized by tumor cells, effector cells will not recognize it as a surface marker.

On the other hand, internalization of antibodies is a favorable effect when a drug or toxin is conjugated to it. Thus, it may be possible to perform a double conjugation to simultaneously link both an immunogenic group (eg, a hapten or polypeptide) and a drug or toxin to an antibody.

In this case, whether the antibody remains on the surface or is incorporated, II
Tumor cells will be damaged. Techniques for attaching drugs and toxins to antibodies are known and described above.

The methods described herein can also be used in conjunction to administer other biological response modifiers to patients. For example, drugs that enhance antigen expression by tumors (e.g., interferons) increase the number of sites available for binding of "engineered antigens" which would actually amplify the immunogenicity of tumor cells. be able to.

The invention will now be illustrated by examples, but the scope of the invention is not limited thereby.

In this example, benzoic acid is conjugated to a monoclonal antibody that recognizes melanoma-associated antigens for the treatment of patients with malignant melanoma.

The antibody used in this example is the murine monoclonal antibody NRX11B, 0, which belongs to the IgG2b subclass.
It is 0. This antibody was generated by fusing mouse tile cells immunized with melanoma proteins with a murine myeloma cell line. This antibody recognizes the 250 kilodalton glycoprotein/proteoglycan melanoma-associated antigen. This antibody can be used as a whole molecule or as a Fab.
Alternatively, it can be used as a Fab' fragment.

Investigation 1: Antibodies (2 to 5 μl/ml) in phosphate buffer (0.0M, pH 5.5) containing 0.1M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (ECDI).
l) to a solution of 20 equivalents of benzoic acid (4/dDSMS
solution) dropwise with vigorous stirring. The reaction mixture is kept at room temperature for 2 hours. At the end of this period, unreacted benzoic acid and ECD I are removed by a disposable size exclusion column (eg, BioRad PD-10 or comparable column). The prepared antibodies are then tested for sterility and safety using standard test methods such as culture, rapid pyrogen test, and limulus test.
ssay) ) 6 This antibody solution is then brought to a suitable concentration for injection and further for use in in vitro immunofenauration procedures.

In this method described, HCD I catalyzes the reaction between the amino groups in the protein molecule and the hydroxyl groups in benzoic acid, thus linking the benzoic acid groups to the antibody. The chemical reaction that occurs is described as follows.

Antibodies Benzoic acid*EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide This method can be performed with other comparable water-soluble carbodiimides.

The patient is a 29-year-old previously healthy man with metastatic malignant melanoma in multiple subcutaneous nodules.

He first undergoes leukopheresis to harvest immune effector cells.

After appropriate expansion in vitro, the antibody/conjugate is given;
Then give the effector cells and then give the lymphokines. These are performed as follows.

FF - Proliferating effector cell populations were described in the literature (Rosenberg,
S.A.

etc., New En Jan Journal of
Medicine, 1985) and expanded upon as follows.

pheresis using a continuous flow cell separator
i s) Obtain a large number of lymphocytes daily by repeating the method. Aim to obtain 5X10'' to 5X10I0 mononuclear cells in each step. Use acid-citrate-dextrose and hevarin as anticoagulants in the transfer bag where the mononuclear cells are collected. The process is repeated for 5 days, with a final volume of 300-400.

Mononuclear cells are separated by Ficoll-Hyberk density gradient. Mix 2-3 parts of Bank's balanced salt solution without calcium or magnesium with 1 part of the leucopheresis cell suspension. 40 ml of diluted cell suspension
Next, cells were incubated at 900X by injecting them into a conical centrifuge tube at 0 d and underlaying lymphocyte separation medium at 10 d.
Centrifuge for 15 minutes at
Resuspend in tissue culture medium containing 2 mM glutamine, 5 M/- gentamicin sulfate, and 2% heat-inactivated human AB serum. A cell prefecture containing 1-10XIO'/d of cells? & Put 11 into 2.51 roller bottle. Recombinant interleukin in these bottles
2 to a final concentration of 1,000 units/d, 0.01
mg/d (l per 1000cc roller bottle)
Add anti-melanoma seclonal antibody/hapten conjugate prepared above a concentration of O■). The roller bottle is incubated at 37° C. by continuous rotation for 3 to 5 days at 0.5 to 1 rpn+. Cells were then incubated in 11 bottles at 500 x g for 15 min, and the pellets were pooled into 250 centrifuge tubes and incubated two more times in Bank's balanced salt solution containing no calcium, magnesium or phenol red. Wash and place in 200d infusion medium containing 0.9% sodium chloride, 5% human normal serum albumin, and 75,000 units of recombinant interleukin-2. Cells are then filtered through a sterile nylon blood dosing filter (110 mesh) and placed in a transfer pack for dosing. Bacterial cultures are obtained at the beginning of cell culture and before administration to the patient, and pyrogen tests are also performed.

The monoclonal antibody conjugate is administered intravenously over 1 hour at a dose of 200 μl in 500 d saline containing 5% human serum albumin. Patients are initially given 1 test dose intravenously and monitored for signs of hypersensitivity reactions during the infusion.

Following administration of antibody, lymphokine (in this case interleukin-2) was administered at 3X10h units/h for 5 consecutive days.
Administer continuously on days. After this infusion begins, activated effector cells are administered daily for 5 days. Cells leukaphereseed and cultured on day 1 are first injected, and each day the next sample is provided in the same order in which they were taken.

Injection of cells begins slowly and is completed in 20-40 minutes if no reaction occurs. For regimen, drugs are administered prior to treatment, such as acetaminophen or indomethacin for fever, meperidine for chills, and other drugs such as anti-emetics and antihistamines.

Antibody/habten administration is repeated at the same dose on the third day of treatment. This also precedes the injection of effector cells. There is no need to interrupt the continuous infusion of IL-2 for antibody administration.

After completing 5 days of treatment, patients have a rest period of 5-10 days before repeating treatment. Subcutaneous nodules are harvested at the cusp of treatment and the localization of antibodies and associated cellular responses are assessed. These observations have predictive value for tumor response. Patients will be followed to assess objective reduction in 111 tumor size. Treatment is repeated in responders unless antiglobulin response is prohibitive. Drugs to suppress the antiglobulin response (
e.g. alkylating agents, steroids, other immunological manipulations)
can be used.

■-On the ground.

Same as Example 1a. However, for all methods (in vitro and in vivo) naked antibodies (with the exception of antibodies with benzoate groups attached) are used.

■-Sat.

Same as Example 1a. However, effector cells are not obtained by cytapheresis but from bone marrow as follows.

The patient is anesthetized and multiple bone marrow aspiration is performed beyond the posterior iliac crest. Collect 400-800 cc of bone marrow and perform serial cell counts as samples are collected to ensure that 3-5 x 10' nucleated cells are obtained at the end of the collection procedure. Filter the cells through a stainless stenol sieve to remove bone marrow debris. The resulting bone marrow suspension is then cultured in roller bottles similar to the technique described in Example 1a for phereseed cells.

The treatment schedule in this example also differs from Example 1a.

Bone marrow cells are cultured for 3-5 days and then treatment begins. After administering the immunoconjugate and IL-2, 30 of the cells obtained
~50% is injected into the patient as described in Example 1a. The remaining cells are kept in culture and the treatment is similarly repeated every 3-5 days for up to 3 weeks. At this point the response is assessed and further treatment decisions made.

In this example, pokeweed antiviral protein, which is a mitogen and a potent antigen, is linked to a monoclonal antibody against a melanoma-associated antigen. This complex is used as a "processed tumor antigen."

5th grade The same antibodies as in Example 1a are used.

The antiviral protein (PAP) is reacted with 10 equivalents of SMCC (succinimidyl 4-[N-areimidomethyl cyclohexacylate carboxylate] as follows: phosphate buffer ( 0,0
A solution of SMCC in DMSO (dimethyl sulfoxide) (4 mg/m
l). After 30 minutes at room temperature, the protein is purified by size exclusion using a column (eg, BioRad PD-10). Add one tenth of DTT (dithiothreitol) to an antibody solution with a concentration of 2 to 5 μ/d in 0.01 M phosphate buffer (pH 7.5) to prepare DTT.
to a final concentration of 25mM. The reaction mixture is held at room temperature for 30 minutes to generate free sulfhydryl groups on the antibody. DTT is removed by size exclusion chromatography. The DTT-treated antibody and SMCC-treated PAP are then immediately mixed and allowed to react for 1 hour. The conjugate is concentrated to approximately 500 ul and injected onto a Sepharose-12 size exclusion column. FP the zygote
LC buffer (0.01M phosphate, 0.5M NaC
1), and both fractions corresponding to the antibody-PAP conjugate are collected and used.

l(■1 translation Same as example 1a.

F-F Same as example 1a.

±-I engineering.

Proceed as in Example 2a. However, in vitro immunization is performed using only PAP (and not the antibody conjugate).

However, the patient will be administered the antibody conjugate and the remainder of the treatment will be as outlined in Example 2a (and la).

劃-J''-0 In this example, a glycosylated monoclonal antibody produced by a recombinant yeast cell after insertion of the relevant gene is used. Since these antibodies contain mannan structures that provide increased immunogenicity, there is no need to use additional haptens, antigens or mitogens. All other aspects of the method are as described in Examples 1a and 2a.

In this example, the class I MHC antigen, LILAA
Alloantigen fragments from 2 or its heavy chain are conjugated to anti-viral antibodies. This immunoconjugate is useful for treating tumors in non-11LA-A2 recipients. HLA-A
Since cytotoxic T cells have been described that react with either the α-1 or α-2 domains of
Existing or ex vivo expanded cytotoxic T-cells can be tested for efficacy in targeting tumors.

1. This protein was developed by Pober et al. (Biochemistry, 20+
5625-32.1981). This can be done using the bifunctional crosslinkers described in the previous examples or by Pierce
Chemical Co (catalog, 1988 2)
Anti-tumor whole 1gG+ (Fab' L or Fab' b fragments) can be linked to the anti-tumor whole 1gG+ (Fab' L or Fab' b fragments) using a cross-linking agent from P. 21-250). This immunoconjugate can be tested for anti-tumor activity.

2. He-A2 11
LA-A2 is a cytotoxic T-lymphocyte (CTL) that has not been precisely defined for any MMC antigen.
) binding site (alloantigenic site) is combined with the known sequence of this antigen (pigueroa and alloantigen site).
(1986) by synthesis of overlapping long peptide fragments. α-1 or α-
Overlapping heavy-duty peptides with a length of 40 or more residues separated by 10 to 20 residues in the primary structure of the two domains, Merrifield et al., Bioche
mistry 215020-31.1983, or Houghtew+, PNAS 82.5131-3
5, 1985 or using a commercially available peptide synthesizer. The peptide, Tam et al.
Cleaved and deprotected from the solid phase resin according to the method of JACS 105.6442-55.1983, 20%
Extracted with acetic acid, lyophilized, and 100% water + 0.1
It can be purified by reverse phase HPLC on a Vydac C-4 analytical column using a 50 minute linear gradient from % trifluoroacetic acid (TFA) to 100% acetonitrile + 0.1% TFA.

PTC-amino acid analysis (Heinrikson et al.
a1. Bioche+a, 136.65-74.19
Peptides are analyzed using 84).

For the anal-shoulder, at least one synthetic peptide will contain residues 98-113 of the heavy (α) chain, which have been shown to block the interaction of CTLs with HLA-A2.

Claims (1)

Claims: 1. A composition for use in a method for eradicating selected target cells in a mammalian host, the composition comprising: sensitized effector cells; an immunogenic molecule comprising an antibody or a conjugate thereof capable of inducing a sensitized effector cell, the immunogen molecule being recognized by the sensitized effector cell, and the sensitized effector cell is homologous to said host,
Said composition. 2. The composition of claim 1, further comprising an effector cell growth factor. 3. A kit for eradicating selected target cells in a mammalian host, comprising two separate compositions, one of which contains sensitized effector cells homologous to the host. and the other composition comprises an immunogenic molecule comprising an antibody or conjugate thereof capable of binding to said target cell, said immunogenic molecule being recognized by said sensitized effector cell. A kit characterized by: 4. The kit of claim 4, further comprising a third separate composition comprising: 4. effector cell growth factor. 5. The composition or kit according to claim 2 or 4, wherein the effector cell growth factor is a lymphokine. 6. The composition or kit according to claim 5, wherein the lymphokine is IL-2. 7. The composition or kit of claim 1 or 3, wherein the immunogenic molecule is an immunoconjugate comprising an immunogenic polypeptide conjugated to a monoclonal antibody that binds to the target cell. 8. The composition or kit of claim 7, wherein the sensitized effector cells recognize the polypeptide. 9. The polypeptide is a non-self human class I or class II histocompatibility antigen, a non-human class I or class II histocompatibility antigen, or a non-human class I or class II histocompatibility antigen.
The composition or kit according to claim 7 or 8, which is an antigen selected from II histocompatibility antigen and alloantigenic domains thereof. 10. The polypeptide is a peptide T-cell epitope, a bacterial cell surface antigen, a viral coat protein,
and immunogenic fragments thereof, according to claim 7 or 8. 11. The composition or kit according to claim 1 or 3, wherein the target cell is a cancer cell. 12. The composition or kit according to claim 1 or 3, wherein the sensitized effector cells are sensitized cytotoxic T-cells. 13, comprising an antibody specific for a target cell to be eradicated in a human or mammalian host, bound to a polypeptide, the polypeptide being of a non-self class.
1. An immunoconjugate selected from the group consisting of a class I or class II histocompatibility antigen, a non-human class I or II histocompatibility antigen, and an alloantigenic domain of the histocompatibility antigen. 14. comprises an antibody specific for the target cell to be eradicated in a human or mammalian host, the polypeptide comprising a peptide T-cell epitope, a cell surface antigen of bacterial origin, a viral coat antigen, and An immunoconjugate selected from the group consisting of immunogenic fragments of. 15. The conjugate according to claim 13 or 14, wherein the target cell is a cancer cell and the antibody is a monoclonal antibody or monoclonal antibody fragment specific for the cancer cell.